Establishment Of Banana CMV And BBTV Detection Methods And Study On Its Content Change

Banana?Musa spp.?is a monocotyledonous plant,belongs to the Musaeae Musa,which is the fourth largest food crops after rice,wheat and corn and one of the largest tropical fruits in the world's fruit consumption and trade.However,large-scale outbreaks and epidemics of Cucumber mosaic virus?CMV?and Banana bunchy top virus?BBTV?have become one of themajor factors limiting the mass production of bananas and have seriously affected the development of the banana industry.Therefore,this study mainly through the establishment of banana CMV and BBTV detection of tissue immunoblotting and clarify the relative changes of the two viruses in different parts of banana and different time,and lays the foundation for the effective prevention and control of disease.In this study,accurate quantification of CMV and BBTV viruses in the leaves of banana samples was carried out by the absolute quantification technique in SYBR Green real-time fluorescence quantitative PCR.By using monoclonal antibodies against CMV and BBTV,Tissue blot immunobiding assay?TBIA?was established for rapid detection of field banana samples.The relative changes of CMV and BBTV in different parts and different periods of bananas were clarified.the results are as follows:1.According to the conserved CP gene sequence of CMV and Rep gene sequence of BBTV,the specific primers of real-time quantitative PCR were designed respectively.A series of gradient dilutions of the cloned positive plasmids were performed and then six dilutions were selected as templates to construct a standard curve,And the rationality of CMV and BBTV real-time quantitative PCR reaction conditions and the reliability of the detection method were determined.The copy number of the CMV contained in the banana sample leaves was 1.10×10¹⁰ copies/?L?1-1??6.84×10⁹ copies/?L?1-2?and 1.00×10¹⁰copies/?L?1-3?,the copy number of BBTV was 4.40×10¹⁰copies/?L?2-1??1.88×10⁸copies/?L?2-2?and 4.17×10⁸copies/?L?2-3?.2.After the study of the TBIA method found that:sensitivity of TBIA detection of banana samples in CMV and BBTV reached the negative three or four times impression,compared with the method of LAMP/RT-LAMP,the sensitivity is not high;O n the stability aspect,the samples were imprinted on the PVDF to save to 4?environment for more than two and a half months can still be stable detect the banana CMV and BBTV,it has good stability.3.The relative content of CMV in different parts was decreased from the upper leaves,the middle leaves to the lower leaves.The relative content of BBTV on leaves were higher than those of the lower leaves and the overall trend was decreasing.In different periods,the relative content of CMV virus in the 8¹2 months and 1 month showed an overall increase first,then declined,and then increased and declined.The relative content of BBTV virus increased first and then decreased.In addition,this study further evaluated the effect of TBIA,LAMP/RT-LAMP and PCR/RT-PCR on the field of banana samples,which was great significance in practical production practice,especially in plant virus control.